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1.
Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.  相似文献   

2.
In budding yeast, Tem1 is a key regulator of mitotic exit. Bfa1/Bub2 stimulates Tem1 GTPase activity as a GTPase-activating protein (GAP). Lte1 possesses a guanine-nucleotide exchange factor (GEF) domain likely for Tem1. However, recent observations showed that cells may control mitotic exit without either Lte1 or Bfa1/Bub2 GAP activity, obscuring how Tem1 is regulated. Here, we assayed BFA1 mutants with varying GAP activities for Tem1, showing for the first time that Bfa1/Bub2 GAP activity inhibits Tem1 in vivo. A decrease in GAP activity allowed cells to bypass mitotic exit defects. Interestingly, different levels of GAP activity were required to prevent mitotic exit depending on the type of perturbation. Although essential, more Bfa1/Bub2 GAP activity was needed for spindle damage than for DNA damage to fully activate the checkpoint. Conversely, Bfa1/Bub2 GAP activity was insufficient to delay mitotic exit in cells with misoriented spindles. Instead, decreased interaction of Bfa1 with Kin4 was observed in BFA1 mutant cells with a defective spindle position checkpoint. These findings demonstrate that there is a GAP-independent surveillance mechanism of Bfa1/Bub2, which, together with the GTP/GDP switch of Tem1, may be required for the genomic stability of cells with misaligned spindles.  相似文献   

3.
The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.  相似文献   

4.
Cell cycle checkpoints can delay mitotic exit in budding yeast. The master controller is the small GTPase Tem1, with inputs from a proposed guanine nucleotide exchange factor (GEF), Lte1, and a GTPase-activating protein (GAP), Bub2/Bfa1. In this issue, Fraschini et al. (p. 335) show that GAP activity of Bub2/Bfa1 appears to be dispensable for inactivation of Tem1 in cells. Their results call into question the GTP/GDP switch model for Tem1 activity, as have other results in the past. The paper also focuses attention on the two spindle pole bodies as potential sites for regulation of Tem1.  相似文献   

5.
ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Deltalte1 mutant and a Deltaras1 Deltaras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.  相似文献   

6.
Kim J  Song K 《Molecules and cells》2006,21(2):251-260
During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit via mitotic checkpoints. In budding yeast, mitotic exit is controlled by a regulatory cascade called the mitotic exit network (MEN). The MEN is regulated by a small GTPase, Tem1p, which in turn is controlled by a two-component GAP, Bfa1p-Bub2p. Recent results suggested that phosphorylation of Bfa1p by the polo-related kinase Cdc5p is also required for triggering mitotic exit, since it decreases the GAP activity of Bfa1p-Bub2p. However, the dispensability of GEF Lte1p for mitotic exit has raised questions about regulation of the MEN by the GTPase activity of Tem1p. We isolated a Bfa1p mutant, Bfa1p(E438K), whose overexpression only partially induced anaphase arrest. The molecular and biochemical functions of Bfa1p(E438K) are similar to those of wild type Bfa1p, except for decreased GAP activity. Interestingly, in BFA1(E438K) cells, the MEN could be regulated with nearly wild type kinetics at physiological temperature, as well as in response to various checkpoint-activating signals, but the cells were more sensitive to spindle damage than wild type. These results suggest that the GAP activity of Bfa1p-Bub2p is responsible for the mitotic arrest caused by spindle damage and Bfa1p overproduction. In addition, the viability of cdc5-2 delta bfa1 cells was not reduced by BFA1(E438K), suggesting that Cdc5p also regulates Bfa1p to activate mitotic exit by other mechanism(s), besides phosphorylation.  相似文献   

7.
Kim J  Luo G  Bahk YY  Song K 《PLoS genetics》2012,8(1):e1002450
In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: (452)S, (453)S, (454)S, and (559)S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa1(4A)) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa1(4D)) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa1(4D) was asymmetrically localized. BFA1(4A) cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA1(4D) cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.  相似文献   

8.
In Saccharomyces cerevisiae, the spindle position checkpoint ensures that cells do not exit mitosis until the mitotic spindle moves into the mother/bud neck and thus guarantees that each cell receives one nucleus [1-6]. Mitotic exit is controlled by the small G protein Tem1p. Tem1p and its GTPase activating protein (GAP) Bub2p/Bfa1p are located on the daughter-bound spindle pole body. The GEF Lte1p is located in the bud. This segregation helps keep Tem1p in its inactive GDP state until the spindle enters the neck. However, the checkpoint functions without Lte1p and apparently senses cytoplasmic microtubules in the mother/bud neck [7-9]. To investigate this mechanism, we examined mutants defective for septins, which compose a ring at the neck [10]. We found that the septin mutants sep7Delta and cdc10Delta are defective in the checkpoint. When movement of the spindle into the neck was delayed, mitotic exit occurred, inappropriately leaving both nuclei in the mother. In sep7Delta and cdc10Delta mutants, Lte1p is mislocalized to the mother. In sep7Delta, but not cdc10Delta, mutants, inappropriate mitotic exit depends on Lte1p. These results suggest that septins serve as a diffusion barrier for Lte1p, and that Cdc10p is needed for the septin ring to serve as a scaffold for a putative microtubule sensor.  相似文献   

9.
The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother‐to‐daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase‐activating protein (GAP) complex Bfa1–Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1–Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1–Bub2 and Tem1 localization at the SPBs. Based on the measured SPB‐bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition.  相似文献   

10.
A role for cell polarity proteins in mitotic exit   总被引:4,自引:0,他引:4  
Höfken T  Schiebel E 《The EMBO journal》2002,21(18):4851-4862
The budding yeast mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis by facilitating the release of the cell cycle phosphatase Cdc14 from the nucleolus. The G protein Tem1 regulates MEN activity. The Tem1 guanine nucleotide exchange factor (GEF) Lte1 associates with the cortex of the bud and activates the MEN upon the formation of an anaphase spindle. Thus, the cell cortex has an important but ill-defined role in MEN regulation. Here, we describe a network of conserved cortical cell polarity proteins that have key roles in mitotic exit. The Rho-like GTPase Cdc42, its GEF Cdc24 and its effector Cla4 [a member of the p21-activated kinases (PAKs)] control the initial binding and activation of Lte1 to the bud cortex. Moreover, Cdc24, Cdc42 and Ste20, another PAK, probably function parallel to Lte1 in facilitating mitotic exit. Finally, the cell polarity proteins Kel1 and Kel2 are present in complexes with both Lte1 and Tem1, and negatively regulate mitotic exit.  相似文献   

11.
The mitotic exit network (MEN) is a spindle pole body (SPB)–associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1–Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2–Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1–Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.  相似文献   

12.
Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2   总被引:3,自引:0,他引:3  
The guanine nucleotide exchange factor Cdc24, the GTPase Cdc42, and the Cdc42 effectors Cla4 and Ste20, two p21-activated kinases, form a signal transduction cascade that promotes mitotic exit in yeast. We performed a genetic screen to identify components of this pathway. Two related bud cortex-associated Cdc42 effectors, Gic1 and Gic2, were obtained as factors that promoted mitotic exit independently of Ste20. The mitotic exit function of Gic1 was dependent on its activation by Cdc42 and on the release of Gic1 from the bud cortex. Gic proteins became essential for mitotic exit when activation of the mitotic exit network through Cdc5 polo kinase and the bud cortex protein Lte1 was impaired. The mitotic exit defect of cdc5-10 Deltalte1 Deltagic1 Deltagic2 cells was rescued by inactivation of the inhibiting Bfa1-Bub2 GTPase-activating protein. Moreover, Gic1 bound directly to Bub2 and prevented binding of the GTPase Tem1 to Bub2. We propose that in anaphase the Cdc42-regulated Gic proteins trigger mitotic exit by interfering with Bfa1-Bub2 GTPase-activating protein function.  相似文献   

13.
The mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis in budding yeast by triggering the nucleolar release and hence activation of the Cdc14 phosphatase. Activation of the MEN is tightly coordinated with spindle position in such a way that Cdc14 is only fully released upon spindle pole body (SPB) migration into the daughter cell. This temporal regulation of the MEN has been proposed to rely in part on the spatial separation of the G-protein Tem1 at the SPB and its nucleotide exchange factor Lte1 confined to the daughter cell cortex. However, the dispensability of LTE1 for survival has raised questions regarding this model. Here using real-time microscopy we show that lte1? mutants not only delay exit from mitosis but also uncouple the normal coordination between spindle disassembly and contraction of the actomyosin ring at cell division. These mitotic defects can be suppressed by a bub2? mutation or by Cdc14 over-expression suggesting that they are caused by compromised MEN activity. Thus Lte1 function is important to fine-tune the timing of mitotic exit and to couple this event with cytokinesis in budding yeast.  相似文献   

14.
Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases.  相似文献   

15.
The elimination of mitotic kinase activity at the end of mitosis is essential for progression to the next stage of the eukaryotic cell cycle. In budding yeast, this process is controlled by a regulatory cascade called the mitotic exit network. Extensive genetic data indicate that mitotic exit network activity is determined by a GTP-binding protein, Tem1, and its putative regulators, Bub2, Bfa1, and Lte1. Here we describe the purification and in vitro activities of Tem1, Bub2, and Bfa1. We describe the nucleotide binding properties of Tem1 and characterize its intrinsic GTPase activity. The combination of Bfa1 and Bub2 acts as a two-component GTPase-activating protein for Tem1. In the absence of Bub2, Bfa1 inhibits the GTPase and GTP exchange activities of Tem1. This inhibition is elicited by either the N- or C-terminal regions of Bfa1, which also retain some ability to co-activate GTPase activity in the presence of Bub2. Although the C-terminal region of Bfa1 binds to Bub2, no interaction of the N-terminal half of Bfa1 with Bub2 was detected despite their combined GAP activity. Therefore, we propose that Bfa1 acts both as an adaptor to connect Bub2 and Tem1 and as an allosteric effector that facilitates this interaction.  相似文献   

16.
In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1–centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.  相似文献   

17.
Yeast spindle pole bodies (SPBs) duplicate once per cell cycle by a conservative mechanism resulting in a pre-existing 'old' and a newly formed SPB. The two SPBs of yeast cells are functionally distinct. It is only the SPB that migrates into the daughter cell, the bud, which carries the Bfa1p-Bub2p GTPase-activating protein (GAP) complex, a component of the spindle positioning checkpoint. We investigated whether the functional difference of the two SPBs correlates with the time of their assembly. We describe that in unperturbed cells the 'old' SPB always migrates into the bud. However, Bfa1p localization is not determined by SPB inheritance. It is the differential interaction of cytoplasmic microtubules with the mother and bud cortex that directs the Bfa1p-Bub2p GAP to the bud-ward-localized SPB. In response to defects of cytoplasmic microtubules to interact with the cell cortex, the Bfa1p-Bub2p complex binds to both SPBs. This may provide a mechanism to delay cell cycle progression when cytoplasmic microtubules fail to orient the spindle. Thus, SPBs are able to sense cytoplasmic microtubule properties and regulate the Bfa1p-Bub2p GAP accordingly.  相似文献   

18.
The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.  相似文献   

19.
The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1-Bub2 guanosine triphosphatase-activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil-like molecule that interacts with the gamma-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.  相似文献   

20.
The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p-Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p-Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.  相似文献   

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